Overexpression of SCC‐S2 correlates with lymph node metastasis and poor prognosis in patients with non‐small‐cell lung cancer

The objective of the current study was to investigate the expression pattern and clinicopathological significance of SCC‐S2 in patients with non‐small‐cell lung cancer (NSCLC). The expression profile of SCC‐S2 in NSCLC tissues and adjacent noncancerous lung tissues was detected by real‐time RT‐PCR, western blot analysis, and immunohistochemistry. In 25 lung cancer tissues examined, 18 (72%) of them exhibited stronger levels of SCC‐S2 mRNA compared with their corresponding normal tissues. SCC‐S2 protein level was up‐regulated in cancerous lung tissues compared to adjacent normal tissue. Moreover, the expression level of SCC‐S2 in 93 archived NSCLC tissues was measured by immunohistochemical staining. SCC‐S2 was found to be overexpressed in 71 of 93 (76.3%) human lung cancer samples and correlated with lymph node metastasis (P = 0.0181), p‐TNM stage (P = 0.0042), Ki‐67 expression (P = 0.0028), and poor survival (P = 0.012). In addition, depleting SCC‐S2 expression by small‐interfering RNA inhibited growth and invasion in lung cell lines. These results indicate that SCC‐S2 plays an important role in NSCLC and might be a useful therapeutic target of NSCLC. (Cancer Sci 2010)     

Therapeutic strategies for afatinib‐resistant lung cancer harboring HER2 alterations

Human epidermal growth factor receptor 2 (HER2) plays an important role in the pathogenesis of various cancers. HER2 alterations have been suggested to be a therapeutic target in non‐small‐cell lung cancer (NSCLC), just as in breast and gastric cancers. We previously reported that the pan‐HER inhibitor afatinib could be a useful therapeutic agent as HER2‐targeted therapy for patients with NSCLC harboring HER2 alterations. However, acquired resistance to afatinib was observed in the clinical setting, similar to the case for other HER inhibitors. Thus, elucidation of the mechanisms underlying the development of acquired drug resistance and exploring means to overcome acquired drug resistance are important issues in the treatment of NSCLC. In this study, we experimentally established afatinib‐resistant cell lines from NSCLC cell lines harboring HER2 alterations, and investigated the mechanisms underlying the acquisition of drug resistance. The established cell lines showed several unique afatinib‐resistance mechanisms, including MET amplification, loss of HER2 amplification and gene expression, epithelial‐to‐mesenchymal transition (EMT) and acquisition of cancer stem cell (CSC)‐like features. The afatinib‐resistant cell lines showing MET amplification were sensitive to the combination of afatinib plus crizotinib (a MET inhibitor), both in vitro and in vivo. The resistant cell lines which showed EMT or had acquired CSC‐like features remained sensitive to docetaxel, like the parental cells. These findings may provide clues to countering the resistance to afatinib in NSCLC patients with HER2 alterations.     

Histone deacetylase inhibitor enhances sensitivity of non‐small‐cell lung cancer cells to 5‐FU/S‐1 via down‐regulation of thymidylate synthase expression and up‐regulation of p21waf1/cip1 expression

It is desirable to find more appropriate therapeutic opportunities in non‐small‐cell lung cancer (NSCLC) due to the current poor prognosis of affected patients. Recently, several histone deacetylase (HDAC) inhibitors, including suberoylanilide hydroxamic acid (SAHA), have been reported to exhibit antitumor activities against NSCLC. S‐1, a novel oral fluorouracil anticancer drug, has been developed for clinical use in the treatment of NSCLC in Japan. Using an MTT assay, we analyzed the growth‐inhibitory effect of 5‐fluorouracil (5‐FU), S‐1, and SAHA against three NSCLC cell lines, as well as the breast cancer cell line MCF7 which is known to be highly sensitive to 5‐FU. Combined treatment with low‐dose SAHA enhanced 5‐FU‐ and S‐1‐mediated cytotoxicity and resulted in synergistic effects, especially in 5‐FU‐resistant cells. Both the mRNA and protein expression levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and orotate phosphoribosyltransferase (OPRT), which are associated with 5‐FU sensitivity/response, were analyzed in the cells undergoing treatment. 5‐Fluorouracil‐resistant lung cancer cells displayed high expression of TS mRNA and protein. Suberoylanilide hydroxamic acid down‐regulated TS mRNA and protein expression, as well as repressed the rapid induction of this factor during 5‐FU treatment, in all examined cell types. We also examined the status of the Rb‐E2F1 pathway, with SAHA up‐regulating p21^waf1/cip1 expression via promoter histone acetylation; this, in turn, blocked the Rb‐E2F1 pathway. We conclude that combination therapy with SAHA and S‐1 in lung cancer may be promising due to its potential to overcome S‐1 resistance via modulation of 5‐FU/S‐1 sensitivity‐associated biomarker (TS) by HDAC inhibitor. (Cancer Sci 2010)     

PAK2通过调节ERK信号通路的活性促进肺癌细胞顺铂耐药

目的:探讨p21激活激酶2(PAK2)在非小细胞肺癌（NSCLC）中是否存在异常表达,以及是否影响肺癌细胞的顺铂敏感性。方法:采用免疫组化和Western blot（WB）方法检测NSCLC中PAK2的蛋白表达模式。利用PAK2-siRNA下调肺癌细胞系中PAK2的表达后,通过WB和细胞功能学实验等方法,明确PAK2对NSCLC细胞系增殖和侵袭能力的影响,并检测顺铂耐药前后的肺癌细胞系中PAK2的表达改变,及其对肺癌细胞顺铂敏感性的影响。结果:PAK2在NSCLC中呈现明显的胞浆强表达,并且其异常表达与肺癌的恶性表型相关;PAK2在顺铂耐药肺癌细胞中表达增强,抑制ERK活性后,PAK2的表达上调不能显著促进肺癌细胞对顺铂耐药。结论:PAK2在NSCLC中发挥着重要的促癌基因功能,并通过调节ERK信号通路的活性介导肺癌细胞系对顺铂耐药性。     

Enhancer of zeste homolog 2 is a novel prognostic biomarker in nonsmall cell lung cancer

BACKGROUND:

Enhancer of zeste homolog 2 (EZH2) epigenetically silences many genes through the trimethylation of histone H3 lysine 27 and is implicated in tumor growth, invasion, and metastasis. However, its role in lung cancer has not been well characterized. The objective of the current study was to elucidate the role of EZH2 in nonsmall cell lung cancer (NSCLC) by investigating both clinical samples and cell lines.

METHODS:

An immunohistochemical analysis of EZH2 expression was performed in samples from patients with stage I NSCLC to investigate the association of EZH2 expression levels with clinicopathologic variables. An in vitro cell growth assay and a Matrigel invasion assay also were conducted in the EZH2‐expressing NSCLC cell lines A549 and H1299 after knocking down EZH2 expression by using an EZH2‐specific short‐hairpin RNA.

RESULTS:

The immunohistochemical analysis classified stage I NSCLC samples (n = 106) into a negative EZH2 expression group (n = 40; 37.7%) and a positive EZH2 expression group (n = 66; 62.3%). Positive EZH2 expression was associated significantly with larger tumor size (P = .014). Kaplan‐Meier survival analyses and log‐rank tests demonstrated that patients whose samples were classified into the positive EZH2 expression group had a significantly shorter overall survival (P = .015). Experiments in the NSCLC cell lines revealed that the knockdown of EZH2 expression reduced the tumor growth rate and invasive activity.

CONCLUSIONS:

The current results indicated that EZH2 promotes progression and invasion of NSCLC, and its expression is a novel prognostic biomarker in NSCLC. Cancer 2011;. © 2011 American Cancer Society.     

MET gene amplification or EGFR mutation activate MET in lung cancers untreated with EGFR tyrosine kinase inhibitors

We analyzed MET protein and copy number in NSCLC with or without EGFR mutations untreated with EGFR tyrosine kinase inhibitors (TKIs). MET copy number was examined in 28 NSCLC and 4 human bronchial epithelial cell lines (HBEC) and 100 primary tumors using quantitative real‐time PCR. Positive results were confirmed by array comparative genomic hybridization and fluorescence in‐situ hybridization. Total and phospho‐MET protein expression was determined in 24 NSCLC and 2 HBEC cell lines using Western blot. EGFR mutations were examined for exon 19 deletions, T790M, and L858R. Knockdown of EGFR with siRNA was performed to examine the relation between EGFR and MET activation. High‐level MET amplification was observed in 3 of 28 NSCLC cell lines and in 2 of 100 primary lung tumors that had not been treated with EGFR‐TKIs. MET protein was highly expressed and phosphorylated in all the 3 cell lines with high MET amplification. In contrast, 6 NSCLC cell lines showed phospho‐MET among 21 NSCLC cell lines without MET amplification (p = 0.042). Furthermore, those 6 cell lines harboring phospho‐MET expression without MET amplification were all EGFR mutant (p = 0.0039). siRNA‐mediated knockdown of EGFR abolished phospho‐MET expression in examined 3 EGFR mutant cell lines of which MET gene copy number was not amplified. By contrast, phospho‐MET expression in 2 cell lines with amplified MET gene was not down‐regulated by knockdown of EGFR. Our results indicated that MET amplification was present in untreated NSCLC and EGFR mutation or MET amplification activated MET protein in NSCLC. © 2008 Wiley‐Liss, Inc.     

The sphingosine kinase 2 inhibitor ABC294640 displays anti‐non‐small cell lung cancer activities in vitro and in vivo

Non‐small cell lung cancer (NSCLC) accounts for about 85‐90% of lung cancer cases, and is the number one killer among cancers in the United States. The majorities of lung cancer patients do not respond well to conventional chemo‐ and/or radio‐therapeutic regimens, and have a dismal 5‐year survival rate of ～15%. The recent introduction of targeted therapy and immunotherapy gives new hopes to NSCLC patients, but even with these agents, not all patients respond, and responses are rarely complete. Thus, there is still an urgent need to identify new therapeutic targets in NSCLC and develop novel anti‐cancer agents. Sphingosine kinase 2 (SphK2) is one of the key enzymes in sphingolipid metabolism. SphK2 expression predicts poor survival in NSCLC patients, and is associated with Gefitinib‐resistance. In this study, the anti‐NSCLC activities of ABC294640, the only first‐in‐class orally available inhibitor of SphK2, were explored. The results obtained indicate that ABC294640 treatment causes significant NSCLC cell apoptosis, cell cycle arrest and suppression of tumor growth in vitro and in vivo. Moreover, lipidomics analyses revealed the complete signature of ceramide and dihydro(dh)‐ceramide species in the NSCLC cell‐lines with or without ABC294640 treatment. These findings indicate that sphingolipid metabolism targeted therapy may be developed as a promising strategy against NSCLC.     

New therapeutic perspectives in CCDC6 deficient lung cancer cells

Non‐small cell lung cancer (NSCLC) is the main cause of cancer‐related death worldwide and new therapeutic strategies are urgently needed. In this study, we have characterized a panel of NSC lung cancer cell lines for the expression of coiled‐coil‐domain containing 6 (CCDC6), a tumor suppressor gene involved in apoptosis and DNA damage response. We show that low CCDC6 protein levels are associated with a weak response to DNA damage and a low number of Rad51 positive foci. Moreover, CCDC6 deficient lung cancer cells show defects in DNA repair via homologous recombination. In accordance with its role in the DNA damage response, CCDC6 attenuation confers resistance to cisplatinum, the current treatment of choice for NSCLC, but sensitizes the cells to olaparib, a small molecule inhibitor of the repair enzymes PARP1/2. Remarkably, the combination of the two drugs is more effective than each agent individually, as demonstrated by a combination index <1. Finally, CCDC6 is expressed at low levels in about 30% of the NSCL tumors we analyzed by TMA immunostaining. The weak CCDC6 protein staining is significatively correlated with the presence of lymph node metastasis (p ≤ 0.02) and negatively correlated to the disease free survival (p ≤ 0.01) and the overall survival (p ≤ 0.05). Collectively, the data indicate that CCDC6 levels provide valuable insight for OS. CCDC6 could represent a predictive biomarker of resistance to conventional single mode therapy and yield insight on tumor sensitivity to PARP inhibitors in NSCLC.     

Novel combination treatment for colorectal cancer using Nek2 siRNA and cisplatin

Nek2 (NIMA‐related kinase 2) is involved in cell division and mitotic regulation by centrosome splitting. We previously reported that Nek2 depletion causes growth suppression and cell death in cholangiocarcinoma and breast cancer cells. In this report, we examine the effect of a combination treatment using Nek2 siRNA with the cytotoxic chemotherapeutic agent cisplatin (CDDP) on colorectal cancer. Nek2 was overexpressed in all colorectal cancer cell lines examined (HCT‐15, DLD‐1, Colo205, and Colo320). Nek2 short‐interfering RNA (siRNA) resulted in the inhibition of cell proliferation and the induction of apoptosis in vitro. Nek2 siRNA suppressed tumor growth compared to control siRNA in a xenograft mouse model. To investigate the potential utility of Nek2 siRNA for clinical cancer therapy, we examine the effect of a combination treatment using Nek2 siRNA with CDDP on colorectal cancer. The combined administration of both Nek2 siRNA and CDDP inhibited cell proliferation and induced apoptotic cell death in vitro. Furthermore, the combined administration of both Nek2 siRNA and CDDP suppressed tumor growth compared to either the single administration of Nek2 siRNA or the combined administration of control siRNA and CDDP. Our results suggest that combination treatment using Nek2 siRNA and chemotherapeutic agents may be an effective therapeutic option for colorectal cancer. (Cancer Sci 2010; 101: 1163–1169)     

O6‐methylguanine DNA methyltransferase repairs platinum‐DNA adducts following cisplatin treatment and predicts prognoses of nasopharyngeal carcinoma

Cisplatin (CDDP) is an important anti‐cancer drug commonly used in various human cancers, including nasopharyngeal carcinoma (NPC). How to overcome the drug resistance of CDDP provides opportunities to improve clinical outcomes of NPC. O⁶‐methylguanine–DNA methyltransferase (MGMT) has been well‐characterized to be a therapeutic determinant of O⁶‐alkylguanine alkylating drugs. However, the underlying mechanism and clinical relevance between MGMT and CDDP remain poorly defined in NPC. In this study, we showed that MGMT‐proficient cells were highly resistant to the cytotoxic effects of CDDP as compared to MGMT‐deficient cells. Further studies showed that the platinum level of DNA after CDDP exposure was significantly lower in MGMT‐proficient cells than in MGMT‐deficient cells. Host cell reactivation assay revealed that MGMT protected NPC cells from CDDP‐induced DNA damage by enhancing DNA repair capacity. Importantly, we demonstrated for the first time that MGMT protein directly bound to CDDP‐induced DNA damages. Subsequently, CDDP‐bound MGMT protein became ubiquitinated and was degraded through ubiquitin‐mediated proteasome system. We further analyzed the relationship between MGMT expression and clinical survivals in a cohort of 83 NPC patients. NPC patients who received CDDP‐based concurrent chemoradiotherapy (CCRT), with high MGMT expression level, exhibited shorter progression‐free survival (PFS; p = 0.022) and overall survival (OS; p = 0.015), than patients with low MGMT expression level. Furthermore, high MGMT expression level remained to be an independent prognostic factor for worse PFS (p = 0.01, hazard ratio 2.23) and OS (p = 0.018, hazard ratio 2.14). Our findings suggest that MGMT protein is important to determine the efficacy of CDDP in NPC.     

Expression of the major vault protein LRP in human non-small-cell lung cancer cells: activation by short-term exposure to antineoplastic drugs

Non-small-cell lung cancer (NSCLC) cells are characterised by resistance to the toxic impact of antineoplastic drugs both in vivo and in vitro. The lung resistance-related protein (LRP), identical with the human major vault protein, is over-expressed in a variety of tumour cells characterised by intrinsic or acquired chemoresistance. We investigated the expression and cellular localisation of LRP in 16 unselected NSCLC cell lines, immortalised bronchial epithelial cells and embryonic lung fibroblasts. All cell lines analysed expressed LRP mRNA, while protein expression ranged from undetectable up to high levels. Cell fractionation and immunofluorescence staining in selected cell lines localised LRP almost exclusively to the cytoplasm. LRP was contained in the 100, 000 g pellet and absent in the soluble, cytosolic fraction and nuclei. A small proportion of LRP, however, was shown to be loosely associated with the outside of the nuclei. Sucrose gradient equilibrium centrifugation revealed presence of LRP exclusively in the fraction known to accumulate purified vault particles. Short-term exposure (16 hr) to subtoxic daunomycin (DM)-, and bleomycin (BM)-concentrations significantly (up to 4-fold) enhanced LRP expression in 2/4 cell lines tested. Cisplatin (CDDP) had a minor effect while vinblastine (VBL) was ineffective. At cytotoxic conditions all drugs rather decreased than increased LRP expression. When basic LRP expression was compared with chemosensitivity, a significant correlation was detected for resistance to CDDP but not DM, doxorubicin (DOX), etoposide (VP-16), VBL and BM. Summing up, our data suggest a role of vaults both in basic CDDP resistance and, additionally, in an short-term defensive response of NSCLC cells against several other drugs.     

Hypoxia‐inducible factor‐1α and nuclear factor‐κB play important roles in regulating programmed cell death ligand 1 expression by epidermal growth factor receptor mutants in non‐small‐cell lung cancer cells

Some driver gene mutations, including epidermal growth factor receptor (EGFR), have been reported to be involved in expression regulation of the immunosuppressive checkpoint protein programmed cell death ligand 1 (PD‐L1), but the underlying mechanism remains obscure. We investigated the potential role and precise mechanism of EGFR mutants in PD‐L1 expression regulation in non‐small‐cell lung cancer (NSCLC) cells. Examination of pivotal EGFR signaling effectors in 8 NSCLC cell lines indicated apparent associations between PD‐L1 overexpression and phosphorylation of AKT and ERK, especially with increased protein levels of phospho‐IκBα (p‐IκBα) and hypoxia‐inducible factor‐1α (HIF‐1α). Flow cytometry results showed stronger membrane co‐expression of EGFR and PD‐L1 in NSCLC cells with EGFR mutants compared with cells carrying WT EGFR. Additionally, ectopic expression or depletion of EGFR mutants and treatment with EGFR pathway inhibitors targeting MEK/ERK, PI3K/AKT, mTOR/S6, IκBα, and HIF‐1α indicated strong accordance among protein levels of PD‐L1, p‐IκBα, and HIF‐1α in NSCLC cells. Further treatment with pathway inhibitors significantly inhibited xenograft tumor growth and p‐IκBα, HIF‐1α, and PD‐L1 expression of NSCLC cells carrying EGFR mutant in nude mice. Moreover, immunohistochemical analysis revealed obviously increased protein levels of p‐IκBα, HIF‐1α, and PD‐L1 in NSCLC tissues with EGFR mutants compared with tissues carrying WT EGFR. Non‐small‐cell lung cancer tissues with either p‐IκBα or HIF‐1α positive staining were more likely to possess elevated PD‐L1 expression compared with tissues scored negative for both p‐IκBα and HIF‐1α. Our findings showed important roles of phosphorylation activation of AKT and ERK and potential interplay and cooperation between NF‐κB and HIF‐1α in PD‐L1 expression regulation by EGFR mutants in NSCLC.     

Loss of YAP1 defines neuroendocrine differentiation of lung tumors

YAP1, the main Hippo pathway effector, is a potent oncogene and is overexpressed in non‐small‐cell lung cancer (NSCLC); however, the YAP1 expression pattern in small‐cell lung cancer (SCLC) has not yet been elucidated in detail. We report that the loss of YAP1 is a special feature of high‐grade neuroendocrine lung tumors. A hierarchical cluster analysis of 15 high‐grade neuroendocrine tumor cell lines containing 14 SCLC cell lines that depended on the genes of Hippo pathway molecules and neuroendocrine markers clearly classified these lines into two groups: the YAP1‐negative and neuroendocrine marker‐positive group (n = 11), and the YAP1‐positive and neuroendocrine marker‐negative group (n = 4). Among the 41 NSCLC cell lines examined, the loss of YAP1 was only observed in one cell line showing the strong expression of neuroendocrine markers. Immunostaining for YAP1, using the sections of 189 NSCLC, 41 SCLC, and 30 large cell neuroendocrine carcinoma (LCNEC) cases, revealed that the loss of YAP1 was common in SCLC (40/41, 98%) and LCNEC (18/30, 60%), but was rare in NSCLC (6/189, 3%). Among the SCLC and LCNEC cases tested, the loss of YAP1 correlated with the expression of neuroendocrine markers, and a survival analysis revealed that YAP1‐negative cases were more chemosensitive than YAP1‐positive cases. Chemosensitivity test for cisplatin using YAP1‐positive/YAP1‐negative SCLC cell lines also showed compatible results. YAP1‐sh‐mediated knockdown induced the neuroendocrine marker RAB3a, which suggested the possible involvement of YAP1 in the regulation of neuroendocrine differentiation. Thus, we showed that the loss of YAP1 has potential as a clinical marker for predicting neuroendocrine features and chemosensitivity.